Leeuwenhoek founded the science of microbiology in about 1700 using a single-lens microscope. His bead of glass had a magnification of 266 times. Fifty years earlier the compound microscope had been discovered. Although Leeuwenhoek's single-lens microscope produced higher resolution and better image quality than the compound microscope, it did not withstand the test of time. The compound microscope, with objective and eyepiece lens systems, became the standard for all modern light microscopes. The principle reason for success of the compound microscope was its ease-of-use. To use the Leeuwenhoek microscope, the sample and microscope had to be placed very close to the eye. This made Leeuwenhoek's simple microscope difficult, and many times nearly impossible, to use. Over the centuries, the compound microscope has been improved and enhanced, but it has largely maintained its use of an objective lens with a relatively long (at least 160 mm) distance to the image formation plane, and an eyepiece to perform second stage magnification and presentation to the human eye.
Lenses are used to create real images in cameras and projectors of many kinds, including video cameras, the part of a television transmitting apparatus that receives the primary image formed by a lens on a light-sensitive material and transforms it into electrical impulses. In its standard use, a video camera transmits images of large objects onto a monitor screen. The image produced by the lens on the light-sensitive material is reduced in size, or demagnified. As objects are brought closer to the lens they appear larger on the monitor screen, but the lens does not create an image larger than the object. Even in a macro mode, the image formed by the lens on the light-sensitive material is not magnified.
A lens creates a magnified image either as a virtual image or as a real image. If an object is placed at a distance closer to the lens than its focal length, then a magnified virtual image is seen when the eye is placed close to the lens. This is the Leeuwenhoek microscope.
If an illuminated object is placed at a distance greater than the focal length of the lens, but less than twice the focal length of the lens, then a magnified real image is projected. The objective lens of the compound light microscope projects a magnified real image. Viewing this real image requires an eyepiece lens or screen. The eyepiece lens magnifies the real image and transforms it into a magnified virtual image seen with the eye. This combination of objective and eyepiece lenses is the compound microscope.
Compound microscopes have been the standard configuration for a light microscope for over two hundred years. They continue to be a primary tool for science and technology. The height of compound microscopes usually exceeds 16 inches and is often much larger. A sample must be positioned on a stage and mechanical devices are required so each person using the microscope can adjust the focus for his/her eyes. People requiring corrective eyeglasses often have difficulty using eyepiece lenses. Special “high eyepoint” eyepieces might be required.
Light microscopes have magnifications that range from 5 to 2,000 times. Most of this magnification is produced by the objective lens that typically has a magnifying power range from 0.5 up to 160 times. The objective lens projects an image 160 mm or more (infinity corrected systems use 180 mm telan lenses to form the primary image) from the objective lens. This projection distance (or tube length) combined with the eyepiece lens is a major factor which establishes the physical size of the microscope. Eyepiece magnifications range from 5 to 20 times, and since the eye must be placed closer to the lens of a high magnification eyepiece, higher magnification eyepieces are difficult to use. A 10 times eyepiece is common, while the majority of light microscopy applications use magnifications between 10 and 500 times.
Video microscopy has been a useful technology for several decades to extend the capability of the compound light microscope. The early uses are documented by Inoue' in Video Microscopy, Plenum Press, New York, 1986. The most common implementation of video microscopy is to attach a video camera to the accessory port on a trinocular viewer. While several types of video cameras were used for video microscopy, the current state-of-art is to use solid-state charge coupled devices (CCD). For low light levels, cooled CCD video cameras are used because of their increased sensitivity. These cameras are very expensive, but have demonstrated unique sensitivity for certain biological applications. The most common uses of video microscopy are to supplement visual observations, record images and provide digital images for image analysis using a digital computer.
When using a compound microscope for photography or video microscopy, a camera apparatus replaces the eye. These cameras contain an additional lens to project a real image onto the film or electronic video-imaging device. Video camera accessories require a special eyepiece lens to reduce the effects of the electronic scaling. In all cases, adding photographic or video cameras increases the size, complexity and cost of the compound light microscope. The addition of a video camera to a compound light microscope can add six to ten inches.
Video cameras have been incorporated into microspectrometer systems for electronic imaging. The system disclosed in U.S. Pat. No. 5,581,085 (Reffner and Wihlborg) used a built-in video camera to generate an electronic image for sample viewing and image recording. Several micro-Raman spectrometer systems use video imaging to protect the viewer from exposure to the laser radiation that is used to generate the Raman spectra. All of these systems use the principles of the compound light microscope with an objective lens, one or more intermediate lenses and a video camera. These video microscope systems use conventional microscope optics with long tube-lengths and add-on video cameras. The standard tube-length is 160 mm (6.3 inch) and the addition of a video imaging system can more than double this length.
Combining microscopes with spectroscopes, spectrometers or spectrographs to provide spectrochemical analysis of small objects or small features of objects has a history of over 125 years (Ford, Single Lens, The Story of the Simple Microscope, Harper & Row, New York, 1985). The early microspectrometers were combinations of visible light microscopes and dispersive spectrometers used for color analysis. In 1949, the first uses of infrared radiant energy in microspectroscopy were reported by Gore, R. C., Science, 110, 70 (1949) and Barer et al, Nature, 163, 198 (1949). In 1953, the first commercial, infrared microspectrometer accessory was produced (Coats et al, J. Opt. Soc. Am, 43, 984, (1953). It was not until the development of Fourier transform infrared (FT-IR) spectrometers that infrared microspectroscopy became a practical technology. The first microscope accessories for FT-IR were introduced in 1983. The design of these accessory microscopes followed the general teachings and design of the compound light microscopy for imaging and spectral measurements.
The wavelength (lambda) of radiation and objective lens aperture limits the spatial resolution of all microscopes. This diffraction limited spatial resolution (d) for a microscope with a limiting numerical aperture (N.A.) is d=(0.62 lambda/N.A.). As the diffraction limited spatial resolution value becomes smaller, the resolving power of the microscope becomes higher. In video microscopy, the resolution element of the photo detector array is an additional factor that can limit resolution. To meet the Nyquest limit, two picture elements are required for a specified resolution to be recorded.
Radiant energy in the mid-infrared range (2.5 to 25 micrometers) is the most useful for the analysis of molecular compounds like organic substances, certain ionic salts and silicate minerals. In this spectral range, the conventional diffraction limited spatial resolution is generally considered to be about 10 micrometers.
To improve spatial definition of the sample area in infrared microspectrometry, Messerschmidt and Sting (U.S. Pat. No. 4,877,960) applied the principles of confocal microscopy. In this development, a pair of image plane masks was used to achieve the confocal geometry as introduced by Minsky (U.S. Pat. No. 3,013,467). Using masks, however, adds complexity and cost to microscopes that are used for infrared microscopical analysis.
Internal reflection microanalysis is achieved by reducing the area of the sample contacting the internal reflection element (IRE) as revealed by Sting and Reffner (U.S. Pat. No. 5,172,182). The teachings of Sting (U.S. Pat. No. 5,093,580) reveals a reflecting ATR microscope objective and a mechanical slider containing aperture masks to select different optical paths through an attached IRE. The ATR objective requires shifting aperture masks between viewing a sample through the IRE and recording its ATR spectrum. By selecting different optical paths, it is possible to collect ATR spectra, observe sample contact or see the sample. While microanalysis with the ATR objective was made easier by these three modes, they added significant cost and complexity.